A novel microfluidic-based fluorescence detection method reveals heavy atom effects on photophysics of fluorophores with high triplet quantum yield: A numerical simulation study

Long lived, dark transient states of fluorescent probes are sensitive to changes in their microenvironment, such as variations in pH, polarity, local oxygen concentration and viscosity. This sensitivity can serve as a biosensing tool allowing researchers to probe and monitor subtle alterations in cellular environments and artificial physiological conditions. The intricate interplay between these states results in transitions that delay fluorescence emission, offering a gateway to phosphorescence-based imaging, particularly for fluorophores with high triplet quantum yields where traditional single molecule detection methods still face major limitations. In the present article, we introduce an idea on a novel fluorescence-based imaging technique combined with a microfluidic platform that enables a precise control of dark transient state populations of fluorescent probes flowing over a uniform, top-flat supergaussian excitation field with a constant flow rate. To demonstrate the imaging capability of the proposed detection method, numerical simulations have been performed by considering laser, microscope and flow parameters of experimental setup together with photophysical model and electronic transition rates of fluorescent dyes. In return, as an output data to be assessed, fluorescence image data is numerically simulated for carboxyfluorescein and its brominated derivative eosin-Y where they both have high triplet and photo-oxidation states with longer lifetime in contrast to rhodamine-based fluorophores. Based on the magnitudes of excitation irradiances and flow rates which can be manually controlled by user during experiments, the presence of dark state populations arising from these emitters can appear as signal broadening, shifts and decays due to pile-up effect in normalized fluorescence intensity signals that are computed from simulated fluorescence images. As such effects become more pronounced for the signals of eosin-Y molecule that contains four heavy bromine atoms, it is elicited that heavy atom effect can be resolved very well by properly tuning excitation power and flow rates. Our proposed imaging modality integrated with microfluidics platform has potential to open up new perspectives in biomedical research where blinking properties of fluorophores can be exploited as a sensor that reflects changes in local environment at the molecular level.